Ultra-High Throughput Screening (Lake Nona)

The Ultra-High-Throughput Screening (uHTS) facility offers the necessary infrastructure for scientists conducting chemical library screening of biological targets. The facility provides diverse screening instrumentation, compound libraries, and expertise in high-throughput screens and automation. The facility supports both user- and facility-driven screening projects. In conjunction with the assay development facilities in Lake Nona and La Jolla, as well as the high-content screening facility, it works to help select and implement the most suitable biochemical or cellular assays for each project.

Provide expertise and advice on high throughput screening approaches
The director of the screening facility has almost 20 years of experience in assay development, high-throughput screening of small molecules/natural products and lab automation. The facility’s staff members are all seasoned scientists, each with greater than 10 years of experience in assay development and HTS in academic or pharmaceutical company settings.

Implement validated assays on automated instrumentation
Once a project has a validated assay (one which is reproducible over a number of days, has a reliable signal to background and has a Z’ of 0.5 or greater) the uHTS facility will implement the assay on our state-of-the-art instrumentation in as high density of plate format that can be used without compromising the performance of the assay. We typically can miniaturize assays to a 1536-well format, which greatly decreases the amount of reagents needed and subsequently the cost of the screen. It is highly recommended that prior to contacting the uHTS facility either the assay aevelopment facilities in Lake Nona, La Jolla, or the high-content screening facility be contacted to obtain help with generating a validated assay for your project.

Provide compound libraries for high throughput screening
There are four library collections available from the Lake Nona uHTS site (see below). More compound collections, mainly smaller sized sets, can be obtained from our compound management facility located at our La Jolla, Calif., site.

Screen validated assays through large compound library sets
Our ultimate goal is to take your validated assay and screen it through one of our large small-molecule compound collections.

uHTS Robotic System

The uHTS facility has at its disposal an industry-class automated system which was integrated by HighRes Biosolutions.

Due to HighRes Biosolution's docking technology, our system is completely modluar. If necessary, we are able to completely reconfigure the system on the fly. Our robotic system is one of the largest and most versatile in the nonprofit arena, with the capability of running almost any type of assay format. The only exception being that we will not run any assays utilizing radioactivity or BSL-3 pathogen. We also have the capability of using all of the instrumentation on the system in either automated or standalone modes.

The uHTS automated system is comprised of three "pods" which contain at their centers a Stäubli RX 160 six-axis industry-class robotic arm. These arms have six degrees of freedom, allowing for optimal flexibility within a spherical work envelop.

The automated system is composed of four classes of peripheral instrumentation that are accessed by the robotic arms:

Plate Storage

Dispensers

Readers

Miscellaneous Instruments

Plate Storage

The system must be able to store all the plates needed to run the assay. This includes both assay as well as compound-library plates. To that end the following storage devices are integrated on our uHTS robotic system:

3x - Liconic LPX440 ambient storage plate carousels. These carousels have the capacity to store up to 440 microtiter plates. Our online compound collection is stored in one of these carousels. Assay plates (both empty or loaded with assay reagents) for assays requiring room temperature incubations are also stored in these carousels.

2x - Liconic STX220 CO2 incubators. These incubators contain a carousel mechanism which allows for the storage of up to 220 microtiter plates. Our STX220s have been modified to accept plates covered with a specialized lid to prevent edge effects commonly seen with cell-based assays.

Dispensers

There are four types of dispensers on our system used to get assay components into the wells of the assay plates. These are:

5x - Labcyte Echo 555 acoustic dispensers. The acoustic dispensers are used to transfer nanoliter quantities of compound from the compound-library plates to the assay plates. They are also used to perform online cherry-picking directly from the same compound-library plate wells that were used in the primary screen. Because the transfer is performed by only using sound waves there is never a chance that cross-contamination of the compounds can occur.

3x - Beckman Coulter BioRAPTR FRD™ dispensers. These instruments, known as bulk dispensers, will dispense a single reagent to all, or most of a plate. They are very precise noncontact dispensers able to dispense up to four seperate reagents to the assay plates.

1x - Beckman Coulter Biomek NX. With its Span-8 bridge, this dispenser allows for transfer of components from one well to another either within the same plate or from one plate to another.

3x - Kalypsys dispenser/washer. These instruments are similar to the BioRAPTRs but with some added benefits. Not only can these instruments work as bulk reagent dispensers, but they also have the capability of washing plates (even 1536-well formats). This capability is essential for running non-homogeneous screens.

Readers

After the assay is finished we need to visualize the results. There are 4 different types of readers available on our system:

3x - PerkinElmer EnVision® Multilabel Plate Reader. This dual-detector PMT-based reader can work with multiple assay technologies including absorbance, ultra-sensitive luminescence, fluorescence intensity, fluorescence polarization, time-resolved fluorescence, and AlphaScreen. All of our readers are equipped with plate stackers that can be added when screens are performed offline.

1x - Hamamatsu FDSS7000. This reader is employed in cell-based real-time kinetic assays. Some of its applications include measuring intracellular Ca2+ flux, membrane potential, ion channel activation, and fluorescence energy resonance transfer. The instrument has the ability to measure in either fluorescence or luminescence modalities.

1x - PerkinElmer ViewLux® uHTS Microplates Imager. This CCD camera-based reader can work with multiple assay technologies including absorbance, ultra-sensitive luminescence, fluorescence intensity, fluorescence polarization, and time-resolved fluorescence. Its advantage over PMT-based readers is that all the wells of an entire plate are read simulatneously, thus the time it takes to read a 1536-well plate is almost the same as a 96-well plate.

Miscellaneous Instruments

The uHTS robotic system has a variety of integrated devices that do not fall into the above categories. These include:

3x - Static 8 position microtiter plate hotels

6x - Plate delidding stations

3x - Barcode scanners

3x - Agilent PlateLoc Thermal Microplate Sealer. This sealer is used to reseal compound plates once the compound has been transferred to the assay plate.

3x - Nexus Biosystems XPeel™ Plate De-Sealer. These automated plate-seal peelers are used to remove the heat seals that are covering the compound plates.

6x - Agilent VSpin® with Access2 automated microtiter plate centrifuge. These centrifuges give us the ability to centrifuge the assay plates and compound plates during the course of a screen.

6x - HighRes Biosolutions MicroBlast units. These instruments are automated deionizing air knife plate cleaners. They help remove any dust and debris that may be present in empty assay plates prior to the addition of assay components or compounds.

Additional Instrumentation

The uHTS facility also has multiple standalone bulk reagent dispensers, liquid handlers and other instrumentation which is used for HTS implementation, unautomated “sneaker” screening, as well as for assay development by the Lake Nona assay development facility. These instruments are:

2x - PerkinElmer Janus® liquid handler. This liquid handler is mainly used by the facility for plate-to-plate transfer, especially for compound library plate creation. It has the ability to work with the following plate types; 96-well, 384-well and 1536-well.

3x - Thermo Multidrop Combi reagent dispenser. This peristaltic pump-based bulk reagent dispenser is capable of dispensing to 96-well, 384-well, and 1536-well plates. It can very rapidly dispense from a range of 0.5 to 2500uL. This dispenser would be akin to our uHTS system's BioRAPTRs.

3x - Thermo Multidrop Combi nL reagent dispenser. This pressure bottle/valve-based bulk reagent dispenser is capable of dispensing to 96-well, 384-well, and 1536-well plates. It can very rapidly dispense from a range of 0.5 to 50uL. This dispenser would be akin to our uHTS system's BioRAPTRs.

1x - BioTek MircoFlo™ Select. This instrument is similar to the Multidrop Combi. It is a peristaltic-based bulk reagent dispenser capable of rapidly dispensing to 96-well, 384-well, and 1536-well plates. This dispenser would be akin to our uHTS system's BioRAPTRs.

1x - Biotek EL406 combination washer dispenser. This washer is set up to wash 1536-well assay plates. It also has a dispenser similar to the MicroFlo above with a slightly angled dispensing head. For the most part it would be used for performing hetergeneous assays, typically those for high-content assays. This dispenser would be akin to our uHTS system's Kalypsys dispenser/washers.

Compound Libraries

The uHTS facility has four compound collections at the Lake Nona site:

SBP 2009 HTS Library:
A large library of approximately 320,000 compounds selected from a pool of over three million compounds from five chemical vendors, ChemBridge, Asinex, Enamine, Life Chemicals, and ChemDiv. They were selected for general HTS screening purpose from a pool of over three million compounds using cheminformatics selection strategies such as a cluster-based 2D-fingerprint approach to ensure diversity balanced with good representation (three to 25 representatives per cluster) and good physicochemical properties (e.g. Lipiski rule of five). Appropriate filters were also applied to exclude compounds with unwanted and reactive groups.

LOPAC collection set:
A collection of 1,280 pharmacologically active compounds from Sigma-Aldrich. This library contains effector molecules of all major target classes, additional details regarding this library are available from Sigma.

The PremiumSet:
This is a collection of 50,000 highly diverse compounds for primary screening from ChemBridge. It is a component of our larger Sanford-Burnham 2009 HTS library, but can be screened on its own instead of the complete 320,000 collection. Additional general information is available from ChemBridge.

We also have the current MLPCN small-molecule collection from the NIH which is composed of approximately 365,000 compounds accessible via the MLPCN NIH roadmap initiative.